The overall aim of this project is to establish an in vitro system to gain insight into the mechanism of IS903 transposition. To this end the IS903 transposase protein will be overexpressed, purified and used in in vitro assays. Assays have been designed to study both intra- and inter-molecular transposition events. In addition, assays to study each of the individual steps in transposition, including DNA binding, synaptic complex formation, DNA cleavage, and integration are described. These assays not only allow the examination of transposition intermediates, they will also allow me to determine the optimal conditions for each step. This information can then be applied to the assays for complete transposition. The IS903 transposase has little similarity to other insertion element transposases; its characterization will provide information as to how apparently unrelated proteins mediate similar events. The IS903 transposon is of particular interest because unlike the majority of other insertion elements it can transpose via two different pathways. A biochemical analysis of the transposition reaction will aid in determining how IS903 achieves this and what determines the choice between the two pathways.